In the art of clinical diagnostics, frequently a diagnostician is faced with the availability of only small sample amounts for testing. Consequently, preparation of samples for testing may result in prepared test solutions having ultralow concentrations of analyte molecules of interest, or the minute amount of available test solution may have an inherent ultralow concentration of analyte to begin with. To address the problem of ultralow concentration of analyte in a test solution, various prior procedures have been developed, such as polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). These conventional techniques relying upon amplification technologies are time consuming and expensive to perform. U.S. Pat. No. 5,866,331 to Singer et al. provides an example of single molecule detection by in situ hybridization techniques.
On the other hand, the ability to detect single molecules in a biomedical application without resorting to amplification techniques has certain advantages, and such measurement techniques without amplification have been developed. For example, molecular beacons, such as taught by Tyagi and Kramer, Nature Biotechnology, Vol. 14, March 1996, pp. 303-308, which bind to target molecules and undergo a conformational change upon binding to specific targets are known. Due to the presence of a fluorophore and quencher, the conformational change in the molecular beacon is translated into an optical signal, which can be detected with a high degree of sensitivity. Furthermore, labeled, constrained DNA smart probes make target labeling and consecutive rinsing steps superfluous in methods for detecting DNA and RNA molecules and fragments.
However, the use of fluorescent probes is limited by certain drawbacks as well. Back-ground signals created by the autofluorescence of the optical fiber, the auto-fluorescence of the solution or solvent in which the analyte of interest is dissolved, or the auto-fluorescence and/or Raleigh and Raman scattering generated by the glass components of the optical measuring system itself can create erroneous readings.
This technique also enhances detection of single molecules in a test solution by employing remote sensing, which allows for combined measurements of free-floating molecular switches in low volume cells as well as for micro-fluidic networks. U.S. Pat. No. 5,814,524 by Walt et al. provides an example of an optical sensor apparatus for optical analytical measurements at remote locations.
In addition, it is possible to chemically attach different molecular switches to several sensor fibers and conduct parallel in situ investigation of several different targets of interest, which reduces the time needed to perform diagnostics. Although setups like this have previously been proposed by Bonnet et al., PNAS, vol. 96, p. 6171, 1999), the principles of signal-collection used by such optical systems are based on the recording of signals that include significant background signals, which severely limits the ultimate detection sensitivity achievable by these methods and apparatuses.
Therefore, there is a need in the art of clinical diagnostics for a sensitive method and apparatus for detecting of low concentrations of analyte molecules that overcomes the drawbacks of the aforementioned methods and apparatuses of the prior art. The present invention endeavors to provide an improved method and apparatus for detecting single analyte molecules in solution that is compatible with, but does not rely upon, conventional amplification techniques, and that is sensitive to the point of being able to detect fluorescence from a single analyte molecule while overcoming the limitations of prior art methods and apparatuses.
Accordingly, a primary object of the present invention is to overcome the disadvantages of the prior art methods and devices for detecting analyte molecules. Another object of the present invention is to provide a method and apparatus for detecting single analyte molecules in solutions that are compatible with amplification techniques, but can be used without them. Another object of the present invention is to provide a method and apparatus for detecting single analyte molecules that is sensitive, easy and convenient to use, and that does not require target labeling and/or consecutive rinsing steps.
Another object of the present invention is to provide a method and apparatus for detecting single analyte molecules that is applicable to miniaturization technologies.
Another object of the present invention is to provide an optical system that performs remote sensing of molecule fluorescence signals and that is especially suited for application to clinical research applications (ex vivo and in vivo), and to practical clinical applications as well.
Another object of the present invention is to provide an optical system that allows in situ measuring of target molecules, thereby making measurements inside the body of a patient possible.